Integrative transcriptome and pigment profiling reveal the molecular mechanism of flower color deepening during the post-flowering stage in chrysanthemum

Flower color is a key trait determining the commercial value of ornamental plants. As changes in flower color are increasingly appealing to home gardening consumers, they have become a key trendsetter for future breeding. However, the molecular mechanism underlying flower color change during the post-flowering stage in flowers remains unclear. In this study, the chrysanthemum cultivar 'f23' (exhibiting deepening flower color with flower aging) was used to explore post-flowering flower color change via an integrated approach of HPLC and transcriptomic analyses. HPLC results showed that two cyanidin glycosides accumulated significantly in ray florets during the post-flowering stage, whereas the total flavonoid content decreased. Transcriptomic analysis revealed that differentially expressed genes were mainly enriched in flavonoid biosynthesis and plant hormone signal transduction pathways. Notably, the ABA signal transduction gene CmSnRK2.6 was identified as a hub gene regulating anthocyanin biosynthesis during the post-flowering stage. Overexpression of CmSnRK2.6 in tobacco resulted in deepened flower color and increased anthocyanin accumulation, accompanied by significant upregulation of core anthocyanin biosynthetic genes. Yeast two-hybrid (Y2H) assays verified a direct physical interaction between CmSnRK2.6 and CmbHLH2.1. Yeast one-hybrid (Y1H) and dual-luciferase assays further confirmed that CmbHLH2.1 directly binds to the promoter of CmDFR and activates its transcription. This study provides a theoretical basis and abundant genetic resources for molecular breeding aimed at flower color improvement in chrysanthemums.